Volume 20, Issue 3, Pages 549-557 (July 2017) CRKL Mediates p110β-Dependent PI3K Signaling in PTEN-Deficient Cancer Cells  Jing Zhang, Xueliang Gao, Fabienne Schmit, Guillaume Adelmant, Michael J. Eck, Jarrod A. Marto, Jean J. Zhao, Thomas M. Roberts  Cell Reports  Volume 20, Issue 3, Pages 549-557 (July 2017) DOI: 10.1016/j.celrep.2017.06.054 Copyright © 2017 The Authors Terms and Conditions

Cell Reports 2017 20, 549-557DOI: (10.1016/j.celrep.2017.06.054) Copyright © 2017 The Authors Terms and Conditions

Figure 1 Identification of CRKL as a Selective p110β-Interacting Protein (A) p110α and p110β differentially associate with binding partners. Proteins reproducibly identified across two independent TAP experiments for p110α and p110β were quantified based on the average intensity of extracted ion chromatograms for the top three most abundant constituent peptides. The color intensity is proportional to the abundance of a protein relative to a given p110 isoform. Gray indicates proteins that were detected but not reliably quantified. White indicates proteins that were not detected. (B) 293FT cells were transduced with BacMam viruses containing His-GST-p85NI, Strep (II)-FLAG-p110α/His-GST-p85NI, or Strep (II)-FLAG-p110β/His-GST-p85NI, followed by immunoprecipitation with anti-FLAG M2 agarose and immunoblot analysis with indicated antibodies. (C) Endogenous CRKL was immunoprecipitated with anti-CRKL antibody from 293FT cell lysates. Rabbit immunoglobulin G (IgG) was used as a control. Cell lysates and immunoprecipitates were analyzed by immunoblotting with indicated antibodies. (D) 293FT cells were cotransfected with FLAG-tagged bovine p85 and HA-tagged wild-type (p110β-WT) or mutant (p110β-Δp85) p110β. Cell lysates were prepared 2 days post-transfection followed by immunoprecipitation with anti-CRKL antibody and immunoblot analysis with indicated antibodies. (E) CRKL requires its SH3N domain to efficiently interact with p110β. Top: schematic illustration of wild-type and mutant CRKL. Bottom: cell lysates from BT549 cells stably expressing LacZ control, V5-tagged wild-type, or mutant human CRKL were immunoprecipitated with anti-V5 antibody and immunoblotted with indicated antibodies. See also Figure S1. Cell Reports 2017 20, 549-557DOI: (10.1016/j.celrep.2017.06.054) Copyright © 2017 The Authors Terms and Conditions

Figure 2 CRKL Knockdown Suppresses PI3K Signaling and Growth of PTEN-Deficient Cancer Cells (A) PC3 and BT549 cells were cultured under a full (10%)-serum condition or under serum starvation overnight. Cell lysates were immunoprecipitated with anti-CRKL antibody or rabbit IgG and immunoblotted with indicated antibodies. (B and C) BT549 (B) and PC3 (C) cells stably transduced with inducible control or CRKL-targeting shRNA were cultured with or without Dox (100 ng/mL, 72 hr) under a full (10%)-serum condition. Alternatively, similarly treated cells were under serum starvation overnight before the harvesting of cells. Cell lysates were analyzed by immunoblotting with CRKL, phospho-AKT (S473), phospho-AKT substrate (RXXS∗/T∗), and tubulin antibodies. The level of pAKT473 was normalized to tubulin. For each experiment, the pAKT473/tubulin ratio in cell lines expressing control shRNA in the absence of Dox was set to 1. Bar graphics reflect the mean ± SEM; n = 3; ∗p < 0.05 (t test). Dox, doxycycline. (D) Stable PC3 cells containing Dox-inducible CRKL shRNA were transduced with LacZ control, shRNA-resistant wild-type (CRKLsilent mutant) or SH3N-domain mutant (CRKLW160L) CRKL cDNAs. Stable cells expressing the respective rescue constructs were cultured with or without Dox (100 ng/mL, 72 hr) under a full (10%)-serum condition. Cell lysates were analyzed by immunoblotting with indicated antibodies. (E) Proliferation of stable PC3 cells containing inducible control or CRKL-targeting shRNA was visualized by crystal violet staining 3 days after Dox (50 ng/mL) induction. Data indicate mean ± SEM; n = 4; ∗p < 0.05 by comparing cells expressing control shRNA and shCRKL (t test). See also Figure S2 and Table S1. Cell Reports 2017 20, 549-557DOI: (10.1016/j.celrep.2017.06.054) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Downregulation of CRKL Does Not Impair p110α-Dependent PI3K Signaling (A) PC3 cells stably transduced with inducible control or CRKL-targeting shRNA were cultured in the presence of doxycycline (Dox; 100 ng/mL, 72 hr). Cells were starved in serum-free medium overnight and treated with indicated inhibitors 1 hr prior to insulin stimulation (4 μg/mL, 10 min). Cell lysates were analyzed by immunoblotting with indicated antibodies. The level of pAKT473 was normalized to tubulin. For each experiment, the pAKT473/tubulin ratio in cell lines expressing control shRNA in the presence of DMSO was set to 1. Bar graphics reflect the mean ± SEM; n = 3; ∗p < 0.05 by comparing cells expressing control shRNA and shCRKL (t test). (B) MCF10A cells stably transduced with inducible control or CRKL-targeting shRNA were cultured with or without Dox (100 ng/mL, 72 hr). Cells were starved in serum-free medium overnight prior to insulin (4 μg/mL, 10 min) or EGF (20 ng/mL, 10 min) stimulation. Cell lysates were analyzed by immunoblotting with the indicated antibodies. The level of pAKT473 was normalized to tubulin. For each experiment, the pAKT473/tubulin ratio in cell line expressing control shRNA in the absence of Dox and stimulated with insulin was set to 1. Bar graphics reflect the mean ± SEM; n = 3. No significant difference was found by comparing cells expressing control shRNA and shCRKL (t test). Cell Reports 2017 20, 549-557DOI: (10.1016/j.celrep.2017.06.054) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Inhibition of PI3K and Src Reduces Cell Proliferation in PTEN-Deficient Cancer Cells (A) PC3, BT549, MDA-MB-361, and MDA-MB-453 cells were cultured under a full (10%)-serum condition or starved in serum-free medium overnight. Cell lysates were immunoprecipitated with anti-CRKL antibody or rabbit IgG and immunoblotted with 4G10, phospho-p130Cas, CRKL, and vinculin antibodies. (B) PC3 and BT549 cells grown under a full (10%)-serum condition or a low (0.1%)-serum condition overnight were treated with PF573228 or dasatinib at indicated concentrations for 10 hr. Cell lysates were immunoblotted with indicated antibodies. (C) PC3 and BT549 cells were cultured under a full (10%)-serum condition or a low (0.1%)-serum condition in the presence of dasatinib and GDC0941 at indicated concentrations. Cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay after 3 days. Data indicate mean ± SD (top left: n = 5; top right: n = 6; bottom left: n = 8; bottom right: n = 8); ∗p < 0.05 (t test). (D) Model representing PI3K signaling under condition of PTEN loss. In cells lacking PTEN, the active FAK-Src complex recruits and phosphorylates the scaffolding protein p130Cas. The phosphorylation of p130Cas creates docking sites for CRKL, thereby recruiting PI3Kβ. The other effectors of CRKL stimulate RAC activity, both a known activator of p110β and a known target downstream of PI3K via PIP3 activated RAC GEFs, forming a potential positive-feedback loop. Cell Reports 2017 20, 549-557DOI: (10.1016/j.celrep.2017.06.054) Copyright © 2017 The Authors Terms and Conditions